Nanoscale Imaging Of Synapses
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Nanoscale Imaging Of Synapses
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Author : U. Valentin Nägerl
language : en
Publisher:
Release Date : 2014
Nanoscale Imaging Of Synapses written by U. Valentin Nägerl and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2014 with Electronic books categories.
Synapses underlie rapid and flexible neural communication in the brain and they hold the key to understanding higher brain functions in health and disease. Because they are very small and highly dynamic, it is very difficult to study them with traditional techniques. Fortunately, recent ground-breaking advances in microscopy have greatly improved our ability to image synapses at the nanoscale, even down to the level of single molecules. Authored by leading practitioners and developers in the field, this volume focuses on the nanoscale analysis of the molecular and structural organization and dynamics of synapses of the central nervous system, utilizing superresolution (e.g. PALM, STORM, STED) and other advanced methods (e.g. EM tomography, optogenetics, FLIM). It explains the basic principles behind the various nanoscale imaging modalities, how they are implemented and what their scope and limitations are, while also highlighting several exciting new research opportunities for synapse research enabled by them.
Nanoscale Imaging Of Synapse Morphology In The Mouse Neocortex In Vivo By Two Photon Sted Microscopy
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Author : Mirelle Jamilla Tamara Ter Veer
language : en
Publisher:
Release Date : 2016
Nanoscale Imaging Of Synapse Morphology In The Mouse Neocortex In Vivo By Two Photon Sted Microscopy written by Mirelle Jamilla Tamara Ter Veer and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2016 with categories.
The brain is a complex organ consisting of neurons and non-neuronal cells. Communication between neurons takes place via synapses, whose morphological remodeling is thought to be crucial for information processing and storage in the mammalian brain. Recently, this neuro-centric view of synaptic function has evolved, also taking into account the glial processes in close vicinity of the synapse. However, as their structure is well below the spatial resolution of conventional light microscopy, progress in investigating them in a physiological environment, the intact brain, has been impeded. Indeed, little is known on the nanoscale morphological variations of dendritic spines, the interaction with glial processes, and how these affect synaptic transmission in vivo. Here, we aim to visualize the dynamic nano-morphology of dendritic spines in mouse somatosensory cortex in vivo. We implemented super-resolution 2P-STED time-lapse imaging, which allows for high spatial resolution and deep tissue penetration, in anesthetized mice, and show that the nano-morphology of spines is diverse, variable, but on average stable, and that differences in spine morphology can have an effect on spine biochemical compartmentalization in vivo. Moreover, implementation of dual color in vivo super-resolution imaging and a novel astrocytic labeling approach provided the first steps towards nanoscale characterization of neuron-glia interactions in vivo. These findings bring new insights in synapse dynamics at the nanoscale in vivo, and our methodological endeavors help pave the way for a better understanding of how nanoscale aspects of spine morphology and their dynamics might contribute to brain physiology and animal behavior.
Quantitative Nanoscale Imaging Of Synaptic Protein Organization
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Author : Lara Laparra Cuervo
language : en
Publisher:
Release Date : 2019
Quantitative Nanoscale Imaging Of Synaptic Protein Organization written by Lara Laparra Cuervo and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2019 with categories.
The arrival of super-resolution techniques has driven researchers to explore biological areas that were unreachable before. Such techniques not only allowed the improvement of spatial resolution in images but also the possibility to perform quantitative measurements at the single-molecule level. The interest in that particular field has been growing over the years and new and more sophisticated tools have been developed. Neuroscience has been one of the first fields to adapt and benefit from super-resolution microscopy. These techniques opened a new window of opportunity to reveal spatial organization of the neuronal cytoskeleton and the molecular organization and dynamics of the synapse, structures below the spatial resolution limit of conventional light microscopy. Protein organization and stoichiometry is central for synaptic transmission in neurons. Knowing the absolute numbers of proteins playing a key role in diseases can be of extreme interest in order to understand the mechanisms that lead to such neurological disorders. In this thesis we exploited the super-resolution techniques to develop a pioneering method for quantitative single-molecule measurements and to unravel the organization of a synaptic protein complex that was never visualized before with nanoscale spatial resolution. We developed a novel method in order to quantify the photoactivation efficiency of eight different photoswitchable fluorescent proteins commonly used in super-resolution experiments. We used the glycine receptor as a template because of its well-known stoichiometry and tagged eight different photoswitchable fluorescent proteins to the a- and ß-subunits of this receptor and transiently transfected them to Xenopus oocytes. The fact that the fluorescent proteins are genetically encoded make them highly suitable for quantitative single-molecule counting. The photoactivation efficiency, which is the percentage of a fluorescent protein that photoactivates into a fluorescently detectable form, plays a critical role in properly interpreting quantitative measurements. Moreover, we also focused our studies on super-resolution imaging of a synaptic protein complex, called LGI1 complex. This ensemble of proteins is one of the main key players involved in different neurological disorders. Leucine rich glioma activated 1 (LGI1) is a neuronal protein that forms a trans-synaptic bridge linking pre- and postsynaptic transmembrane proteins (ADAM22 and ADAM23) and helps to organize a multimeric complex at the synapse including AMPA receptors and voltage-gated potassium channels (VGKC). LGI1 autoimmune encephalitis is a severe neuropsychiatric disorder related to epilepsy where the patients produce autoantibodies against LGI1, which alter synaptic plasticity. However, the molecular mechanisms that lead to the observed problems in patients still remain largely unknown. Using well-characterized synaptic markers as molecular standards, we determined the positioning of LGI1 and the other related proteins within the synaptic space at nanoscale resolution by means of multi-color STORM. Further, the comparison of this molecular architecture in healthy neurons versus neurons treated with antibodies from patients suffering from LGI1 autoimmune encephalitis showed that these antibodies impact the nanoscale organization of pre-synaptic proteins. These results suggested a loss of LGI1 interaction with pre-synaptic proteins upon antibody binding and gave further insight into early changes in pathology.
Sted Nanoscopy Of Synaptic Substructures In Living Mice
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Author : Jennifer-Magdalena Masch
language : en
Publisher:
Release Date : 2017
Sted Nanoscopy Of Synaptic Substructures In Living Mice written by Jennifer-Magdalena Masch and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2017 with categories.
Optical nanoscopy has revolutionized far-field microscopy, enabling the observation of subcellular structures and dynamics from completely new perspectives. Among other fields, neuroscience benefits greatly from the remarkable advances in super-resolution microscopy, which provide unprecedented insights into the molecular organization and function of synapses. STED nanoscopy has been one of the most successful methods for live-cell applications and is the only superresolution technique that has been demonstrated for imaging in living mice so far. However, in vivo nanoscale imaging of synapt...
Expansion Microscopy Of C Elegans
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Author : Chih-Chieh Jay Yu
language : en
Publisher:
Release Date : 2020
Expansion Microscopy Of C Elegans written by Chih-Chieh Jay Yu and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020 with categories.
Expansion microscopy (ExM) enables 3-D, nanoscale-precise imaging of biological specimens by isotropic swelling of hydrogel-embedded, chemically processed tissue. Such capability raises the question of whether nanoscale mapping of biomolecules could be performed in an entire organism, which would allow super-resolution-mediated in situ analyses, such as digital quantification of biomolecules and mapping of synaptic contacts, to be performed within the context of an entire nervous system. The nematode Caenorhabditis elegans could be a suitable model for such organism-wide analyses, due to its tractable physical size, deterministic cell lineage, ease of genetic control, and well-established literature. However, C. elegans is enclosed in a chemically impermeable and mechanically tough cuticle, which could hinder the deployment of ExM. In this thesis, we present a strategy, expansion of C. elegans (ExCel), to expand fixed, cuticle-enclosed intact animals of C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNAs, DNA locations, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins at a ~25-nm resolution (20x linear expansion). We demonstrate the utility of the ExCel toolbox for multiplexed imaging of multiple molecular types, for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal. In addition to ExCel, we discuss two other ExM-related technologies, including tetragel, which is a highly homogeneous hydrogel network that improves the nanoscale isotropy of biological ultrastructure expanded by ExM, and stochastic arrangement of reporters in clusters (STARC), which is a strategy for recording neuronal activity at a subneurite-level resolution, in densely labeled neuronal populations. Taken together, the work presented in this thesis extends the capabilities of ExM, and lays the foundation for a comprehensive, functionally and structurally informed analysis of an entire organism, which could reveal new insights in neuroscience, organismal development, and systems biology.
Far Field Optical Nanoscopy
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Author : Philip Tinnefeld
language : en
Publisher: Springer
Release Date : 2015-02-07
Far Field Optical Nanoscopy written by Philip Tinnefeld and has been published by Springer this book supported file pdf, txt, epub, kindle and other format this book has been release on 2015-02-07 with Science categories.
This book describes developments in the field of super-resolution fluorescence microscopy or nanoscopy. In 11 chapters, distinguished scientists and leaders in their respective fields describe different nanoscopy approaches, various labeling technologies, and concrete applications. The topics covered include the principles and applications of the most popular nanoscopy techniques STED and (f)PALM/STORM, along with advances brought about by fluorescent proteins and organic dyes optimized for fluorescence nanoscopy. Furthermore, the photophysics of fluorescent labels is addressed, specifically for improving their photoswitching capabilities. Important applications are also discussed, such as the tracking and counting of molecules to determine acting forces in cells, and quantitative cellular imaging, respectively, as well as the mapping of chemical reaction centers at the nano-scale. The 2014 Chemistry Nobel Prize® was awarded for the ground-breaking developments of super-resolved fluorescence microscopy. In this book, which was co-edited by one of the prize winners, readers will find the most recent developments in this field.
Single Molecule Microscopy In Neurobiology
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Author : Nobuhiko Yamamoto
language : en
Publisher: Humana
Release Date : 2021-05-13
Single Molecule Microscopy In Neurobiology written by Nobuhiko Yamamoto and has been published by Humana this book supported file pdf, txt, epub, kindle and other format this book has been release on 2021-05-13 with Medical categories.
This volume looks at the methodology and techniques used by experts to study how certain molecules function in specific locations, and their temporal patterns. Chapters in this book cover topics such as in vivo single-molecule tracking of voltage-gated calcium channels with split-fluorescent proteins in CRISPR-engineering C. elegans; protein-protein interactions in membranes using single particle tracking; neuropathological diseases revealed by quantum-dot single particle tracking; SPoD-OnSPAN; and investigating molecular diffusion inside small neuronal compartments with two-photon fluorescence correlation spectroscopy. In the Neuromethods series style, chapters include the kind of detail and key advice from the specialists needed to get successful results in your laboratory. Cutting-edge and comprehensive, Single Molecule Microscopy is a valuable resource for any researcher interested in learning more about this important field.
Imaging Synapse Structure And Function
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Author : George J. Augustine
language : en
Publisher: Frontiers Media SA
Release Date : 2017-07-06
Imaging Synapse Structure And Function written by George J. Augustine and has been published by Frontiers Media SA this book supported file pdf, txt, epub, kindle and other format this book has been release on 2017-07-06 with Electronic book categories.
Development of new imaging technologies in recent years has transformed neuroscience in profound ways. Following on the heels of the revolution based on the Green Fluorescent Protein, refined genetically-encoded fluorescent reporters and genetic targeting strategies now enable optical recording of synaptic transmission in defined neuronal populations at speeds approaching the enviable temporal resolution of electrophysiology. Super-resolution light microscopy permits observation of synapses and their molecular machinery at sub-diffraction resolution. At the ultrastructural level, automated forms of electron microscopy, improvements in specimen fixation methods, and recent efforts to correlate data from light and electron micrographs now make the reconstruction of functional neural circuits a reality. Finally, the use of optogenetic actuators, such as channelrhodopsins, allows precise temporal and spatial manipulation of neuronal activity and is revealing profound insights into the organization of neural circuits and their roles in behavior. This research topic highlights recent advances in both light and electron microscopy, with a specific focus on approaches that combine innovations from several different fields to obtain novel information about synapse structure and function. We are confident that this collection of articles - three original research papers, six reviews, one methods paper and one perspective article - will enable neuroscientists to achieve the next generation of experiments aimed at cracking the neural code.
Pharmacology Of Neurotransmitter Release
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Author : Thomas C. Südhof
language : en
Publisher: Springer Science & Business Media
Release Date : 2007-12-07
Pharmacology Of Neurotransmitter Release written by Thomas C. Südhof and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2007-12-07 with Medical categories.
It has been known for half a century that neurotransmitters are released in preformed quanta, that the quanta represent transmitter-storing vesicles, and that release occurs by exocytosis. The focus of this book is twofold. In the first part, the molecular events of exocytosis are analysed. In the second part of the book, the presynaptic receptors for endogenous chemical signals are presented that make neurotransmitter release a highly regulated process.
Nanoscale Photonic Imaging
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Author : Tim Salditt
language : en
Publisher: Springer Nature
Release Date : 2020-06-09
Nanoscale Photonic Imaging written by Tim Salditt and has been published by Springer Nature this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020-06-09 with Science categories.
This open access book, edited and authored by a team of world-leading researchers, provides a broad overview of advanced photonic methods for nanoscale visualization, as well as describing a range of fascinating in-depth studies. Introductory chapters cover the most relevant physics and basic methods that young researchers need to master in order to work effectively in the field of nanoscale photonic imaging, from physical first principles, to instrumentation, to mathematical foundations of imaging and data analysis. Subsequent chapters demonstrate how these cutting edge methods are applied to a variety of systems, including complex fluids and biomolecular systems, for visualizing their structure and dynamics, in space and on timescales extending over many orders of magnitude down to the femtosecond range. Progress in nanoscale photonic imaging in Göttingen has been the sum total of more than a decade of work by a wide range of scientists and mathematicians across disciplines, working together in a vibrant collaboration of a kind rarely matched. This volume presents the highlights of their research achievements and serves as a record of the unique and remarkable constellation of contributors, as well as looking ahead at the future prospects in this field. It will serve not only as a useful reference for experienced researchers but also as a valuable point of entry for newcomers.